Project Summary Epigenetics regulates gene expression without changing nucleotide sequence. DNA methylation and histone modification are two major mechanisms in epigenetic control. Development and cellular differentiation are epigenetic changes in vertebrates. Changes in DNA methylation state accompany various stage of development, from oocyte implantation blastocyst formation, and from stem cells to many differentiated cell types. DNA methylation is also associated with genetic imprinting, X-chromosome inactivation, and transposon silencing. Because it is broadly involved in gene regulation, epigenetics plays an increasingly recognized important role in cancer development, neurological disorder such as RETT syndrome, aging, diabetes, obesity, heart disease and response to environmental factors. The dynamics of DNA methylation requires cytosine methylation and demethylation. In mammals, cytosine demthylation is a complicated process requires the participation of two proteins. 5-methylcytosine (mC) is oxidized by TET (ten eleven-translocation protein) to 5- hydroxymethylC (hmC), 5-formylC (fC), 5-carboxylC (caC) in a Fe2+- and 2-oxoglutarate-dependent manner. fC and caC are then removed by TDG (thymine DNA glycosylase) and replaced by unmethylated cytosine via base excision repair (BER) pathway. TDG belongs to family 2 in uracil DNA glycosylase (UDG) superfamily. We identified an unusual TDG that is robust and specific for the caC base through bioinformatics search and biochemical characterization. Our central hypothesis is that this new TDG adopts a unique catalytic mechanism that underlies its base recognition specificity and caC excision. The objective of this application is to use an integrated biochemical, enzyme kinetics, structural biology approach coupled with computational mutual information analysis to define the molecular mechanism underlying specificity and catalysis in this enzyme and human TDG. The successful execution of the research plan will provide valuable insight on how this enzyme has evolved into a caC DNA glycosylase, open new avenues for understanding specificity in TDG, and lay the foundation for engineering TDG with tailored specificity for in vitro and in vivo epigenetics investigation.